Screening for Novel LOX and SOD1 Variants in Keratoconus Patients from Brazil

Abstract

Purpose: To investigate the presence of the variants of lysyl oxygenase (LOX) and superoxide dismutase 1 (SOD1) genes in Brazilian patients with advanced keratoconus.

Methods: Donor genomic DNA extracted from blood samples was screened for 5'UTR, exonic LOX, and SOD1 variants in a subset of 26 patients presenting with advanced keratoconus (KISA $>$ 1000% and I-S $>$ 2.0) by Sanger sequencing. The impact of non-synonymous amino acid changes was evaluated by SIFT, PMUT, and PolyPhen algorithms. The Mutation Taster tool was used to evaluate the potential impact of formation of new donor and acceptor splice sites in the promoter region of affected volunteers carrying sequence variants. A 7-base SOD1 deletion (IVS2 + 50del7bp) previously associated with keratoconus was screened in 140 patients presenting classical keratoconus by gel fragment analysis, and positive samples were sequenced for confirmation.

Results: We found an unreported missense variant in LOX exon 6 in one heterozygous patient, leading to substitution of proline with threonine at residue 392 (p. Thr392Pro) of LOX protein sequence. This mutation was predicted to be potentially damaging to LOX protein. Another LOX variant, Arg158Gln, was also detected in another patient but predicted to be non-pathogenic. Two additional new polymorphisms in LOX 5'UTR region (-116C $>$ T and -58C $>$ T) were found in two patients presenting with advanced keratoconus and were predicted to modulate or create donor/acceptor splice sites in LOX transcripts. Additionally, SOD1 deletion was detected in one patient presenting with severe keratoconus, not in control samples.

Conclusion: We described three novel LOX polymorphisms identified for the first time in Brazilian patients with advanced keratoconus, as well as a previously described SOD1 deletion strongly associated with keratoconus. A possible role of these variants in modulating transcript levels in the cornea of affected individual requires further investigation.

Keywords: LOX; Mutation; SOD1; Keratoconus.

Figure 1

Electropherograms showing R158Q (c.773 C $>$ T) (A) and T392P (c.1474T $>$ A) (B) variants. Genotypes are indicated, and dotted vertical lines indicate peak location for each alteration. Sequencing of T392P variant was obtained using reverse primer.

Figure 2

Conservation of LOX primary amino acid sequence in several species. Positions of residues R158 and T392 relative to Homo sapiens LOX protein are indicated.

Figure 3

Detection of the IVS2 + 50del7bp deletion in SOD1 gene in one patient affected with advanced keratoconus. (A) PAGE electrophoresis showing a subset of patients tested for the deletion (numbers indicated above). Amplicons of 218 pb (normal) and 211 pb (deleted) fragments are indicated by black arrows. A 1 kb DNA ladder shows the 300 pb reference fragment (smaller fragments are not shown). (B) Electropherograms of the deletion region from normal and affected patients. (C) Corneal topographies of KC patient carrying the SOD1 deletion. PB, base pairs; KB, kilobases; OD, right eye; OE, left eye.

Figure 4

Electropherograms showing 5'UTR polymorphisms located at positions –116C $>$ T (A) and –58C $>$ T (B) relative to SOD1 ATG start site. Sequencing was performed using reverse primer.

Figure 5

DNA sequence alignment of SOD1 5'UTR sequences from different species. Conserved positions of residues –116 and –58 relative to Homo sapiens sequence are indicated.